These studies are designed to clarify the role of metabolic activation in the toxicity and carcinogenicity of phenacetin. This problem will be approached through analysis of enzyme activities, nucleic and protein binding, mutagenicity and carcinogenicity studies. Enzyme activities to be assayed include: N-O-acyltransfer, O-deethylation, N-hydroxylation, deacetylation and glucuronide and sulfate conjugation. Of special interest will be the N-hydroxylation of phenacetin and further activation of N-hydroxyphenacetin by acyltransfer, sulfate conjugation, or other pathways. Potential target tissues from rat and human sources will be used in these studies. Phenacetin and N-hydroxy-phenacetin labeled in various positions will allow analysis of metabolic pathways in macromolecular binding studies. In addition to in vitro studies, the carcinogenicity of phenacetin will be assessed. Attempts will be made to alter the carcinogenic potential of phenacetin through metabolic manipulation and promotion by saccharin. These experiments should provide insight into the molecular mechanisms involved and the potential synergistic effects of these commonly encountered agents.